Tumor suppressor genes and oncogenes play an important role in the regulation of cell growth, differentiation and apoptosis. These proteins can either be growth stimulatory or growth inhibitory, depending on cell type, growth conditions or differentiation state. We have developed a sensitive in vitro transformation assay to measure transformation of NIH 3T3 cells. NIH 3T3 cells are made competent for transforming by transfection with the ras oncogene. The activity of the ras-gene can then be enhanced or blocked by oncogene transfection of the human genes c-fos and c-myc, respectively. Oncogene transfection alters the transformed phenotype, which becomes morphologically flat, epitheloid, less adherent and is selected for by growing the cells in soft agar. By increasing or decreasing oncogene expression in NIH 3T3 cells, we were able to alter the transformed phenotype. The oncogene transfected cells are tested for their ability to form colonies in soft agar under selective conditions. Cells with transformed phenotypes grow well in soft agar under selective conditions while control cells (empty vector transfected) fail to grow. We are using this transformation assay to screen small molecule inhibitors of cell growth for antitumor activity. The goal is to identify and test inhibitors of the c-myc oncogene for antitumor activity. The c-myc oncogene is overexpressed in about 10% of human cancers. We screened about 80 small molecules for their ability to selectively inhibit c-myc induced tumor cell growth in the absence of cytotoxic effects. In addition to the antitumor activity, tumor selectivity was the most important criteria for identifying potential antitumor drugs. Thus, we identified a number of compounds that are active in the cell growth assay and inactive in the cytotoxic assay. We then characterized one compound in detail. In addition, we identified additional potential antitumor compounds that remain to be fully evaluated. We have used the cell culture assay to identify tumor-selective compounds with activity against the c-myc and other oncogenes, and we are continuing to screen additional compounds in order to find more selective antitumor drugs. There are currently about 2000 small molecules that have been tested as potential inhibitors of cell growth or inducers of apoptosis. We identified 12 compounds that inhibit c-myc-induced transformation with IC50 values in the range of 15 to 40 nM and were identified as high priority drug candidates. The mechanism of action of these compounds has been studied extensively. The c-myc responsive promoter has been shown to be required for transformation, suggesting the possibility that these compounds directly act on transcription of the c-myc gene. Since we can now show in cell culture that inhibition of c-myc oncogene expression can lead to tumor cell growth arrest and apoptosis, these compounds should be considered for in vivo antitumor testing and may represent novel therapeutics. The molecular targets for these antitumor compounds remain unknown. We have also used c-myc-induced transformation as a model for studying oncogene regulation in response to physiological stress. Our focus has been on bcl-xl, a member of the Bcl-2 family of apoptosis regulators. We have demonstrated that c-myc is necessary for bcl-xl-induced apoptosis in NIH 3T3 cells. However, bcl-xl expression in normal proliferating tissues is insufficient for mediating apoptosis. In addition, there is no induction of apoptosis in primary cells under conditions of stress, but the cells still express bcl-xl. Thus, we have demonstrated that expression of bcl-xl is not sufficient for apoptosis even when present in excess. We have been using bcl-xl as a model of another member of the Bcl-2 family of apoptosis regulators. The expression of bcl-xl in a cancer cell line appears to block induction of apoptosis by death receptor ligation. We are currently determining the mechanism of inhibition of cell death by bcl-xl in a cell culture model. We are also comparing gene expression in breast cancers containing mutant p53 with bcl-xl mutations to tumors lacking mutations in both genes. It is also important to understand why cells do not undergo apoptosis when bcl-xl is expressed in large amounts. A portion of this research is aimed at defining small molecule inhibitors of bcl-xl to determine the function of bcl-xl in cell death and also how the interaction of bcl-xl and bax leads to cell death. If we understand this interaction and can block it in cancer cells, this would have implications for cancer treatment. We have found that cells with the normal form of bax will undergo apoptosis when treated with the anticancer drug etoposide, however, bax mutants cells do not undergo apoptosis when treated with this drug. We are currently determining the molecular mechanism by which bax can function as a tumor suppressor. While we are characterizing the bcl-xl mutants, we have also found that these mutations are dominant, suggesting that they interfere with the function of normal bax or other bcl-2 proteins. In particular, the dominant mutations are in regions that make bax insoluble and prevent it from interacting with bcl-2 family proteins. Thus, we have been characterizing these dominant negative mutants. Our initial results suggest that cells containing the dominant negative mutations are resistant to the drug etoposide. Since etoposide is a topoisomerase II inhibitor, this suggests that the DNA damage induced by topoisomerase II inhibitors leads to activation of bax. The bax protein may be required for the cell death response to certain anticancer drugs. We are now examining whether bax functions as a tumor suppressor and how etoposide induces apoptosis. To this end, we have purified recombinant bax protein and are studying the interaction of this protein with bcl-2 family members in order to characterize the mechanism of apoptosis induced by etoposide. If we can define the function of bax, we should have a better understanding of the regulation of cell death and be able to intervene more effectively in this process. We are using molecular and cellular biology approaches for the study of apoptosis regulators. Some of these approaches require transfection of genes into mammalian cells. Transfection of genes into mammalian cells is achieved by methods that include calcium phosphate precipitation, cationic lipids, DEAE-dextran, electroporation, microinjection, and viral transduction. We use a combination of approaches that include calcium phosphate precipitation and cationic lipids for the transfection of DNA into mammalian cells. Calcium phosphate precipitation is the easiest method and does not require the use of special equipment. Transfection of DNA into mammalian cells by calcium phosphate precipitation has been a widely used technique for many years. We began using calcium phosphate precipitation over 20 years ago, and it is a popular method in our laboratory. In this procedure, DNA and cells are combined in a phosphate-buffered saline solution containing calcium chloride, and the transfection is completed by incubation at 37xc2x0 C. for 1 hour. We have used calcium phosphate transfection to transfect a variety of plasmids, including luciferase reporter plasmids, at different cell densities. To demonstrate the validity of this approach in our laboratory, we first optimized this method for the transfection of plasmids into COS-1 and HeLa cells using transfection vectors, including pSV2CAT, pCAGGS, and pcDNA3, which have been widely used in our laboratory for a variety of transfection experiments. The results demonstrate the validity of this